1. To identify key differentiation genes and characterize their molecular mechanisms of action A. Identification, Cloning and Characterization of CASZ1, the human homolog of the drosophila neural fate determination gene castor (dCas). We have identified, cloned and characterized CASZ1, the human homolog of the drosophila neural fate determination gene, dCas. We determined it mapped to chromosome 1p36.22, and CASZ1 expression increased during retinoid differentiation. In drosophila, dCas is expressed exclusively at a late stage of neuroblast development that precedes the cessation of proliferation and the beginning of neuronal differentiation. The stage at which castor is expressed during drosophila development is analogous to later stages of human embryonic neural organogenesis, which implies that the human homologue- may have the same role as dCas in controlling cell fates within neuroblast cell lineages. Building on our previous studies that structurally characterized human CASZ1 genes and expression in a number of differentiation models, we embarked on a functional study of CASZ1 in mammalian cells. We generated a series of tet-regulated CASZ1 expressing cell lines;4 neuroblastoma cell lines and 1 rhabdomyosarcoma cell lines. Using unbiased microarray analyses we identified putative CASZ1 targets were over-represented in functional pathways associated with muscle and neural differentiation, cell motility and cell growth. Functional studies in neuroblastoma indicate it suppresses colony formation, tumor cell growth induces differentiation. Increased CASZ1 expression suppresses tumor growth in pre-clinical animal models. Previously we had identified a number of siRNAs to target the MYCN gene. We have developed vector systems that can over-express and turn-off MYCN. These have been invaluable in our studies to identify MYCN targets involved in cell cycle regulation and differentiation. In a series of collaborative studies we have contributed to studies that identified that MYCN expression in tumors stimulates recruitment of natural killer cells to tumors. Our studies also contributed to the recent identification that the candidate tumor suppressor microRNA miR-34a directly regulates the MYCN gene. Both these studies have importance as they increase our understanding as to why NB tumors that have over-expression of MYCN have a more aggressive phenotype and patients have the worst overall survival. C. The Cancer Stem Cell hypothesis proposes that a rare cell in the tumor, which is highly resistant to current therapies, is responsible for repopulating the tumor while the progeny of this Cancer stem cell, which are responsible for the bulk of the tumor, are relatively more sensitive to the therapies. This theory would explain why tumors relapse. We developed a unique orthotopic model for neuroblastoma that enabled us to assess the tumor initiating capacity of tumors. Contrary to the Cancer Stem Cell hypothesis we find that 1/10 cell in a primary culture of neuroblastoma cells and 1/2 cells from a neuroblastoma cell lines is capable of initiating a tumor. This indicates that the almost every cell in a NB tumor has tumor initiating capacity. Ongoing studies are evaluating whether environmental cues cause changes in epigenetic regulation of developmental programs in these cells.